* To be published in Insectes Sociaux, in press
A
BSTRACTThe remarkable ability of slave-making ants to integrate chemically in the colonies of their host species makes them useful model systems for investigating the role of cuticular hydrocarbons in chemical recognition. The purpose of our study was to examine the influence of the rearing host species on the cuticular hydrocarbon profile and on the mating behaviour of sexuals of the slave-making ant Chalepoxenus muellerianus. Sexuals from a population parasitizing exclusively the host species Temnothorax unifasciatus were reared in the laboratory either with their natural host or another potential host species, Temnothorax recedens. C. muellerianus males reared with T. unifasciatus investigated and mounted female sexuals reared with the same host significantly more often than female sexuals reared with T. recedens. Similarly, C. muellerianus males reared with T. unifasciatus discriminated against female sexuals from natural T. recedens colonies. Males experimentally or naturally reared with T. recedens did not clearly discriminate between female sexuals reared by the two host species and only rarely engaged in mating attempts with either type of female sexuals.
Chemical analyses showed that host species affect the chemical profile of C. muellerianus sexuals and vice versa. Our findings indicate that cuticular hydrocarbons might be important in the mating success of this ant species.
Keywords: Chalepoxenus muellerianus, social parasitism, mating success, cuticular hydro-carbons, Formicoxenini
I
NTRODUCTIONIndividuals in sexually reproducing species need to locate and recognize appropriate mating partners and therefore have to collect information about their potential mates’ species, gender, and quality. Social Hymenoptera (bees, ants, and wasps) are no exception, though sex plays a role only during a very limited period of their lives and is therefore rarely studied (Boomsma et al., 2005). Sexual communication in ants is mostly mediated by volatile pheromones, which are produced in the mandibular glands of the males of some species and in various glands in the female sexuals of others and serve to attract mating partners from a distance (reviewed by Ayasse et al., 2001). Ant males intensively antennate female sexuals before mating (e.g.
Hölldobler and Bartz, 1985), suggesting that additional signals are involved in short-range communication. Cuticular hydrocarbons, a blend of surface chemicals known to be crucially important in nestmate recognition and other aspects of interindividual communication (e.g., Soroker et al., 1994, 1995; Vander Meer and Morel, 1998; Lahav et al., 1999; D'Ettorre and Heinze, 2005), might provide such signals. In many insect species, cuticular hydrocarbons play indeed a fundamental role in the context of mating, e.g., the recognition of sex (Carlson et al., 1984; Ginzel et al., 2003; Howard, 1998), mate choice (Howard et al., 2003), and as sex pheromones and male attractants (Blomquist et al., 1993, 1998; Ayasse and Dutzler, 1998;
Ayasse et al., 1999; Schiestl et al., 1999). However, detailed experiments on near-range com-munication among ant sexuals are lacking.
The special lifestyle of slave-making ants and the way in which they are chemically integrated in the colonies of their hosts allows investigating, which role cuticular hydrocarbons play in courtship and mating (D'Ettorre and Heinze, 2001). Slave-making ants are social parasites that depend on the help of host workers from other ant species for all routine tasks in their nests. For the cohesion of their mixed colonies it is necessary that slavemakers and hosts possess a common colony odour (Lenoir et al., 2001). It has previously been shown that the cuticular hydrocarbon profiles of slave-making ants closely resemble those of their hosts, probably because slavemakers synthesize the host chemicals and/or acquire cuticular hydrocarbons from their hosts through grooming and other interactions (Franks et al., 1990; Yamaoka, 1990; Bonavita-Cougourdan et al., 1997, 2004; D'Ettorre et al., 2002; Kaib et al., 1993; Lenoir et al., 2001). Slave-making ants are therefore a particularly useful model system for studying interactions between social environment, chemical signature, and behaviour.
The slave-making ant Chalepoxenus muellerianus (Finzi, 1922) enslaves 12 or more species of the related genus Temnothorax. In contrast to several other formicoxenine slavemakers (Buschinger, 1966; Buschinger and Alloway, 1977; Buschinger and Winter, 1983), colonies and also whole populations of C. muellerianus typically parasitize only a single of several suitable host species present. For example, 96.6% of all C. muellerianus colonies investigated by Buschinger et al. (1988a) contained only one of several sympatric Temnothorax host species. About 3/4 of the colonies utilised exclusively T. unifasciatus (Latreille, 1798), 10% contained T. recedens (Nylander, 1856), and only 3.4% had a mixture of several host species. Schumann and Buschinger (1994, 1995) suggested that this preference for a single host species results from imprinting of young slavemakers on the odour of the host present in their nests: for colony foundation, young parasite queens preferentially invade colonies of the host species they are familiar with and likewise, during slave raids slavemaker workers pillage brood only of the species already present as hosts in their maternal colonies.
The importance of imprinting and early learning of olfactory stimuli in host choice has previously been demonstrated also in the Nearctic slave-making ant Polyergus lucidus (Goodloe and Sanwald, 1985; Goodloe et al. 1987), and imprinting appears also to be important in other contexts, such as brood acceptance (Jaisson, 1975; Le Moli and Passetti, 1977; Le Moli and Mori, 1982) and habitat choice (e.g., Jaisson, 1980; Dijeto-Lordon and Dejean, 1999).
The chemical basis for the imprinting effect in C. muellerianus is unknown, neither for workers nor for sexuals. It has also not yet been studied whether imprinting affects mate choice and in this way might eventually lead to the formation of host races. Goodloe et al.
(1987) already suggested determining whether mate choice by slavemaker sexuals, in particular those of Polyergus lucidus, is influenced by the host species in their partner's nest of origin. As cuticular hydrocarbons play an important role in interindividual recognition and in the mating process of many insect species, we investigated how the host species affects the cuticular hydrocarbon profile of C. muellerianus male and female sexuals and conducted mating experiments with slavemaker sexuals reared with two different host species, T. unifasciatus and T. recedens.
M
ATERIAL ANDM
ETHODSAnt collecting and culture
In March 2003, colonies of the myrmicine slavemaker Chalepoxenus muellerianus and the two host species Temnothorax unifasciatus and T. recedens were collected from dry walls and rotten sticks at Tignale and Rovato at Lago di Garda, Italy. In these populations, C. muellerianus exclusively utilizes T. unifasciatus as host, and mixed colonies only contained the slavemaker and T. unifasciatus slaves. T. recedens, which is used as host species by C. muellerianus in other populations, occurs in the same sites and with the same density as T. unifasciatus (Schumann and Buschinger, 1994). In late April 2005, Chalepoxenus muellerianus colonies were collected near Baška on the island of Krk, Croatia.
Colonies from Baška use only T. recedens as host species and T. unifasciatus was not found at this site.
The ants were housed in the laboratory in artificial nests in plastic boxes (10 x 10 x 3 cm³) with three connected chambers and a regularly moistened plaster floor (Buschinger, 1974; Heinze and Ortius, 1991). Twice a week all colonies were fed with diluted honey and pieces of cockroaches. Ants were kept in an incubator under semi-natural conditions with an annual cycle of artificial seasons with daily temperature variations and a natural photoperiod.
Behavioural experiments Rearing of sexuals
Of the 23 C. muellerianus laboratory colonies from Italy with T. unifasciatus as host, 65%
produced sexual pupae (a total of 146 female sexuals and 117 males in 2003, 45 female sexuals and 167 males in 2004, and 24 female sexuals and 66 males in 2005). As C. muellerianus sexuals reared with T. recedens were not available from the field in 2003 and 2004, about half of the unpigmented, young male and female pupae were removed from their mother colonies and transferred into free-living Italian T. recedens colonies (n = 19). Pupae were used because they can easily be transferred between nests of different ant species (Schumann and Buschinger, 1991, 1994; D'Ettorre et al., 2002; Errard et al., 2006a).
C. muellerianus sexuals eclosed about three weeks later. After a short period, the callows start to synthesize their own cuticular hydrocarbons and to adsorb chemicals from their nestmates to integrate into the colony (Lenoir et al., 2001; Morel and Blum, 1988; Dahbi et al., 1998).
We thus obtained male and female sexuals of Italian C. muellerianus that had either eclosed
in nests of their original host T. unifasciatus or in T. recedens nests. Female sexuals of C. muellerianus reared by T. unifasciatus workers are hereafter referred to as FU, female sexuals reared by T. recedens workers as FR. Similarly, C. muellerianus males reared by T. unifasciatus are referred to as MU and males reared by T. recedens workers as MR.
Additionally, of the 15 C. muellerianus colonies from Croatia with T. recedens as original host species, 53% produced sexual pupae in 2005 (a total of 62 female sexuals and 48 males). C. muellerianus female sexuals from Krk are referred to as FRK, males as MRK.
C. muellerianus from Krk could not be transferred into sympatric T. unifasciatus colonies, as this species was not found at the collecting site.
Mating experiments
Behavioural experiments were carried out after eclosion of the sexuals between June and August 2003, between July and September 2004, and in August and September 2005, when the ants experienced summer conditions (28°C, 12 h light and 17°C, 12 h dark). Though there are no reports about the mating behaviour of Chalepoxenus in the field, it is known that young sexuals, as in most formicoxenine ants, leave the nest in summer to engage in mating flights (Buschinger et al., 1988b). In the field, winged and dealate C. muellerianus females have been collected in July and August 1986 near Rome (Mei, 1992). When ready to mate, young Chalepoxenus sexuals leave their nests about 2 weeks after eclosion and crawl or flutter around (Schumann and Buschinger, 1994; Buschinger et al., 1988b). Though female sexuals do not perform any visible sexual calling behaviour, they appear to produce sexual pheromones in the poison gland, which attract males (Buschinger et al., 1988b).
A total of 132 behavioural experiments were conducted in plastic flight cages of 35 x 22 x 29 cm³ in 2003, and 24 x 16 x 17 cm³ in 2004 and 2005 with a wire mesh on two sides to allow an air flow (Table 4-1). Trials were carried out on a meadow at the university campus at Regensburg or in the laboratory when the weather was cold and unstable. Since swarming behaviour naturally occurs between 4 and 9 pm (Schumann and Buschinger, 1994), the ex-periments were carried out in the afternoon, mostly around 2 to 8 pm. Only sexuals that had left the nest chambers of the formicaries were used for the mating experiments. For each trial, one female and one male C. muellerianus sexual were placed together in a flight cage.
Although C. muellerianus form loose mating swarms in the field, only two individuals were used per trial, because marking these small, winged ants individually would have had a negative effect on their condition and presumably influenced mating behaviour. Females and males inside a flight cage were unrelated and each ant was used only in one experiment. The
frequency of the social interactions between each pair was recorded for 90 to 150 minutes, depending on the weather conditions. However, most of the trials (88%) lasted 120 minutes.
The duration of the trials did not influence the activity of sexuals.
As complete mating sequences are often difficult to obtain in flight cages, we in particular focussed on the gentle, inspective antennation of the other individual’s body, which typically precedes copulation attempts and in C. muellerianus reliably occurred in flight cages, and on mating attempts, i.e., males trying to mount the female sexuals and to insert their genitalia into the female cloaca. Complete copulations, during which the male tilted backwards with inserted genitalia, were rarely observed.
Behavioural data were analysed by Mann-Whitney U-tests, χ²-tests, and, in the case of smaller data sets, two-sample permutation tests. The occurrence of antennations differed between 2003 and 2004, probably due to differences in cage size and/or environmental conditions (Mann-Whitney U-test: antennal contacts/min performed, males: U = 93.0;
p = 0.0001; female sexuals: U = 176.0; p = 0.0001) and data from the two years were there-fore analysed separately. The number of mating attempts/min did not differ between 2003 and 2004 (U = 458.5; p = 0.382) and therefore could be pooled for a χ²-test. Data from 2005 with C. muellerianus, which naturally parasitized T. recedens, were again analysed separately.
Combined probabilities were obtained using Stouffer’s method, which is superior to Fisher’s combined probability test (e.g., Whitlock, 2005).
Chemical analysis
We investigated the cuticular hydrocarbons of 100 C. muellerianus sexuals from ten T. recedens and ten T. unifasciatus colonies (26 FU; 21 FR; 33 MU; 20 MR), 10 slaves each from two colonies with T. unifasciatus (SU) and two with T. recedens (SR), and 10 workers each from three unparasitized T. unifasciatus (WU) and three unparasitized T. recedens (WR) colonies from Lago di Garda. Ants were killed by freezing and cuticular compounds were extracted by immersing them individually in 25 µl of pentane for 15 min. After evaporation of the solvent, the residues were re-dissolved in 10 µl of pentane and 2 µl of this solution were then injected into an Agilent Technologies 6890N gas chromatograph equipped with a flame ionisation detector and a capillary column. The injector was split-splitless, and the carrying gas was helium at 1ml/min. For half of the samples, a Rtx-5 capillary column (30 m x 0.25 mm x 0.50 µm, Restek, Bellefonte, USA) and a temperature program of 1 min at 80°C, from 80°C to 180°C at 30°C/min, from 180°C to 280°C at 4°C/min, and then held at 280°C for 22 min was used. The other half of the samples was analysed on a HP-5 capillary column
(30 m x 0.32 mm x 0.25 µm, J&W Scientific, USA) with a temperature program of 1 min at 80°C, from 80°C to 180°C at 30°C/min, from 180°C to 300°C at 4°C/min, and then held at 300°C for 12 min. The resulting chromatograms obtained with both methods were comparable and yielded the same pattern of peaks. Gas chromatography gave consistently 33 peaks, of which 21 could be identified by comparison with standards and from their mass spectra, produced by electron ionisation mass spectrometry using a Hewlett Packard (Palo Alto, CA, USA) 5890A gas chromatograph coupled to an HP 5917A mass selective detector.
The analysis was performed on a HP-5MS column (30 m x 0.247 mm x 0.25 µm) with a temperature program of 1 min at 80°C, from 80°C to 180°C at 30°C/min, from 180°C to 280°C at 4°C/min, and then held at 280°C for 22 min. Electron impact mass spectra were obtained with an ionisation voltage of 70eV and a source temperature of 230°C.
Chemical profiles were compared by multivariate statistical analysis (Statistica 6.0, Statsoft, and SPSS 13.0, SPSS Inc.). The proportions of the 33 consistently detectable peaks were first analysed by principal component analysis (PCA) to reduce the number of variables used in the following discriminant analysis (DA). The standardized discriminant function coefficients and the factor loadings (> 0.7) were used to assess the importance of single compounds. DA was used to determine whether defined groups could be distinguished on the basis of their cuticular profiles and to assess the degree of similarity between groups. The correct classification of individuals to the respective groups was verified. Groups were also compared by calculating the squared Mahalanobis distances between the group centroids.
R
ESULTSBehaviour
We investigated whether the species of the host that cared for the eclosing C. muellerianus sexuals had an influence on their interactions during courtship and mating. In particular, we analysed the number of antennal contacts per minute of males towards female sexuals and vice versa. Results for male sexuals are shown in Fig. 4-1 (a, b: 2003; c, d: 2004). Male and female sexuals did not differ in their antennation rates in 2003 (two-sample permutation test, nFU = 18, nMU = 23, p = 0.212; nFR = 17, nMR = 12, p = 0.378), but males were significantly more active in 2004 (nFU = 14, nMU = 13, p = 0.022; nFR = 16, nMR = 17, p = 0.007). Although there was a significant difference in the antennation rate of MU and MR towards FU in 2003 (nMU = 12, nMR = 6, p = 0.043), differences in activity were only marginally significant in 2004 (nMU = 6, nMR = 8, p = 0.080) and not significant concerning the antennation of MU and MR towards FR (2003: nMU = 11, nMR = 6, p = 0.140; 2004: nMU = 7, nMR = 9, p = 0.904).
Males reared by a particular host seemed to be more interested in female sexuals reared by the same host than those reared by the other host. In 2003, MU antennated FU significantly more often than FR (nFU = 12, nFR = 11, p = 0.027; Fig. 4-1a), but a difference in the same direction was not significant in the second year with a smaller sample size (nFU = 6, nFR = 7, p = 0.799; Fig. 4-1c). Similarly, in both years the average antennation rate of MR towards FR was higher than towards FU, though not significantly so (2003: nFU = nFR = 6, p = 0.228, Fig. 4-1b; 2004: nFU = 8, nFR = 9, p = 0.087, Fig. 4-1d). In 2005, MU also antennated FU significantly more often than female sexuals from natural colonies from Krk with T. recedens as slaves (nFU = 16, nFRK = 20, p = 0.002; Fig. 4-1e). The antennation rate of MRK did not differ between FRK and FU (nFU = 29, nFRK = 2, p = 0.477 Fig. 4-1f). Taking the results from the three years together, MU males appeared to be significantly more attracted to virgin queens reared by T. unifasciatus (Stouffer’s method, ZS = -2.292, p = 0.0017), while males reared by T. recedens did not choose (ZS = -1.247, p = 0.107).
Female sexuals did not preferentially contact a certain type of male (FU, 2003: nMU = 12, nMR = 6, p = 0.427; 2004: nMU = 6, nMR = 8, p = 0.146; 2005: nMU = 16, nMRK = 2, p = 0.727, ZS = -0.367, p = 0.358; FR, 2003: nMU = 11, nMR = 6, p = 0.686; 2004: nMU = 7, nMR = 9, p = 0.362, FRK: nMU = 20, nMRK = 29, p = 0.628, ZS = 0.262, p = 0.603).
Figure 4-1. Antennal contacts per minute performed by male sexuals. a, b: 2003. c, d: 2004. e, f: 2005.
In the upper left corner the actor is indicated, the receiver is given below the bars. Sample size is given below. Box plots show the median (), 25% and 75% quartiles and minimum and maximum values.
Significance levels from permutation tests are indicated as follows: ** < 0.01; * p < 0.05, + 0.05 < p <
0.1, NS p > 0.1
Summed over 2003 and 2004, significantly more MU-FU pairs than MU-FR pairs showed mating activity, while there was no significant difference between MR-FR and MR-FU pairs. Only FU-MU pairs succeeded in copulating (Table 4-1). Concerning mating activity, neither MU nor MRK showed a significant preference for a certain kind of female (χ²-test, MU: χ² = 2.56, p = 0.109; MRK: χ² = 1.44, p = 0.230; Table 4-1). Again, the only pair that succeeded in copulating consisted of a male and a female sexual reared with T. unifasciatus (Table 4-1).
Table 4-1. Number of trials, number of successful copulations, and number of pairs of the slave-making ant Chalepoxenus muellerianus showing mating activity ; FU stands for C. muellerianus female sexuals reared by T. unifasciatus, FR for C. muellerianus female sexuals reared by T. recedens, and MU and MR are C. muellerianus males reared by T. unifasciatus and T. recedens, respectively.
FRK and MRK are T. recedens reared sexuals from Krk.
Chemical analysis
C. muellerianus sexuals and their host workers show a complex cuticular profile characterized by linear and methyl-branched alkanes with chain lengths from C25 to C31 (Fig. 4-2). PCA performed on the 33 compounds of C. muellerianus sexuals, their T. unifasciatus (SU) and T. recedens slaves (SR) and workers from unparasitized host colonies (WU, WR) produced 8 principal components with eigenvalues greater than 1, explaining 76% of the total variance. A subsequent discriminant analysis performed on 9 variables (factor loading > 0.7) significantly separated the eight groups (Wilks’ λ = 0.0013, F(56,678) = 29.53078, p < 0.0001; Fig. 4-3) and correctly classified 87.1% of all individuals. The classification was particularly good for MU (93.9%), FR (95.2%), SU (90%), SR (100%), WU (90%) and WR (90%), while only 84.6%
of FU and 60% of MR were correctly classified. Two of 26 FU were classified each as MU and FR, and of the 20 MR four were classified as MU, two as SU, and one each as FU and FR. Function 1 accounted for 67%, function 2 for 18% of the total variance.
Male Female Number
Figure 4-2. Representative gas-chromatograms of female sexuals (F) and males (M) of Chalepoxenus muellerianus reared with Temnothorax unifasciatus (U) and with Temnothorax recedens (R), and of T. unifasciatus (SU) and T. recedens (SR) slaves, with indication of the identified peaks: (1) C25; (2) 11-Me-C25 +13-Me-C25; (3) 5-Me-C25; (4) 3-Me-C25; (5) C26; (6) 4-Me-C26; (7) C27; (8) 11-Me-C27 +13-Me-C27; (9) 7-Me-C27 +5-Me-C27; (10) 3-Me-C27; (11) C28; (12) 2-Me-C28 +4-Me-C28; (13) C29; (14) 11-Me-C29 + 13-Me-C29 + 15-Me-C29; (15) 7-Me-C29; (16) 11,15-diMe-C29 + 13,17-diMe-C29; (17) 3-Me-C29; (18) 5,y-diMe-C29; (19) C31; (20) x-Me-C31; (21) 5-Me-C31.
All squared Mahalanobis distances between the eight groups were statistically significant. The distance between C. muellerianus sexuals and SU was always smaller than that between C. muellerianus and SR. This means that the hydrocarbon pattern of the C. muellerianus sexuals was more similar to T. unifasciatus slaves than to T. recedens slaves.
However, sexuals reared with T. unifasciatus clearly differed from those reared with T. recedens, i.e., the rearing host species influenced the chemical profile of Chalepoxenus.
Comparing the distance between FU/FR and MU/MR, the influence of T. recedens appeared to be stronger in Chalepoxenus female sexuals than in males, because in females the difference was larger. Furthermore, slaves were considerably more similar to C. muellerianus than to unparasitized workers, suggesting that they acquire substances from their parasites.
Interestingly, the distance between workers from unparasitized colonies of T. unifasciatus and T. recedens was considerably smaller than that between parasitized workers of both species, i.e., cuticular profiles of unparasitized workers of both host species were much more similar to each other than the profiles of slaves from both host species (Table 4-2).